Hepatitis C virus (HCV) is the major etiological agent of 90% of all cases of non-A, non-B hepatitis (Dymock, B. W. Emerging Drugs 6:13–42 (2001)). The incidence of HCV infection is becoming an increasingly severe public health concern with 2–15% individuals infected worldwide. While primary infection with HCV is often asymptomatic, most HCV infections progress to a chronic state that can persist for decades. Of those with chronic HCV infections, it is believed that about 20–50% will eventually develop chronic liver disease (e.g. cirrhosis) and 20–30% of these cases will lead to liver failure or liver cancer. As the current HCV-infected population ages, the morbidity and mortality associated with HCV are expected to triple.
Known treatments for HCV infection include the use of interferon-α (IFN), which indirectly effects HCV infection by stimulating the host antiviral response. IFN treatment is largely ineffective, however, as a sustained antiviral response is produced in less than 30% of treated patients. Further, IFN treatment induces an array of side effects of varying severity in upwards of 90% of patients (e.g. acute pancreatitis, depression, retinopathy, thyroiditis). Therapy with a combination of IFN and ribavirin has provided a slightly higher sustained response rate, but has not alleviated the IFN-induced side effects.
One research area of active interest includes the development of antiviral agents which inactivate virally encoded protein products essential for HCV viral replication. Examples of such agents include various tripeptide compounds, which act as selective HCV NS3 serine protease inhibitors. However, many of these compounds do not sufficiently inhibit HCV protease activity or do not have sufficient potency, and thus, may not provide optimal treatment of HCV-infected patients. Accordingly, there is an ongoing need for the development of HCV assays for the identification of agents effective for inactivating viral replication proteins.
Known cell-based assays for screening compounds for HCV inhibitory activity rely upon the detection of viral RNA replication using RT-PCR (Ito et al., Hepatology 34(3):566–572 (2001); Bartenschlager R. and V. Lohman, Antiviral Res. 52(1):1–17 (2001)). Such cell-based systems often yield variable results, making reproducibility a major problem and the use of such system for the screening of compounds impractical, particularly for use in high throughput screening (HTS). HCV assays which rely on the inhibition of viral enzymes essential for viral replication and which may be suitable for HTS are known (Bianchi et al., Analytical Biochemistry 237, 239–244 (1996); Taliani et al., Analytical Biochemistry 240, 60–67 (1996)), but such assays measure only in vitro activity.
Accordingly, there exists a need for an accurate and reproducible cell-based HCV assays which permits the screening of compounds for HCV replication inhibitory activity. The present invention is directed towards such assays.